PROJECT SUMMARY We developed an innovative set of phenotypic in vivo assays to discover drugs that slow neurodegeneration in Alzheimer's disease (AD) and frontotemporal dementia (FTD), the most common age-related dementias . The high-throughput screening (HTS) assays are based on an automated imaging technology, robotic microscopy (RM), that we invented that longitudinally tracks individual cells in complex mixtures of cell types. With fluores- cent biosensors, RM can measure almost any biological event in a single live neuron over its lifetime. Uniquely, RM automates imaging of the same neurons and microscope fields repetitively and can collect lifetime information on individual neurons. The HTS assays we developed will be used to discover new drugs to treat neurodegenerative diseases by testing for their effects in the CNS of zebrafish AD models. Because zebrafish larvae are optically transparent, they are amenable to the longitudinal imaging analysis afforded by RM. Our primary HTS assay will measure the effects of drugs on the turnover of tau in single neurons in zebrafish larvae CNS. Tau is an integral part of our assays because it is a key protein in AD and FTD, and its overexpression is associated with the buildup of toxic phosphorylated tau (p-tau), which is linked to neurodegeneration in these diseases. Small-molecule drugs that increase clearance of p-tau should slow neurodegeneration and progression in AD and FTD. To monitor tau turnover in single brain neurons, we integrated into RM, optical pulse-labeling (OPL), an imaging technology that monitors the lifetime of a protein in a single cell. We showed that OPL can monitor mutant and wild-type tau turnover in neurons in vivo in zebrafish. Incorporated into our primary HTS is a follow-up assay to quantify changes in p-tau levels in the same neuron we measure tau turnover. To optimize the HTS nature of the primary assay, we will screen it against a small- molecule proteostasis library to establish the sensitivity of the assay to identify compounds that increase tau clearance. The primary assay will then be transferred to the SMDC at UCSF for HTS of a 10,000-compound small-molecular library to identify those compounds that increase tau turnover in CNS in vivo. Hits from this HTS program will be evaluated by us for efficacy in reducing p-tau levels in CNS and for potency. Hits will then be tested by us in a secondary screening assay to monitor activity of neurons in larvae in vivo with RM and the Ca2+ biosensor CaMPARI. This assay will allow us to prioritize compounds that increase tau clearance and prevent tau disruption of circuit activity and that do not produce non-selective effects on CNS activity that could cause side effects in clinical use. Effective compounds will then be tested in an additional secondary assay for efficacy in blocking tau-mediated neurodegeneration in zebrafish by a novel RM technology that incorporate the highly sensitive GEDI probe. These studies will identify compounds that block tau-mediated neurodegeneration in vivo whose mechanism of action is to increase tau and p-tau clearance that can be further developed to treat AD.